We will attempt to elucidate the major mechanisms of hyperbaric oxygen (HBO) damage in mitochondria and to perform preliminary experiments to correlate results with HBO damage in rats. The hypothesis is that increased superoxide production associated with HBO treatment leads to a cascade of events that resembles previously characterized effects of tert-butyl hydroperoxide (tBH) and other thiol oxidizing reagents, but that superoxide is a much more potent reagent for protein-thiol oxidation that tBH is. The hypothesis will be tested by an analysis of the comparative effects of progressive HBO and tBH treatment in a BSA model system and in mitochondria. Earlier tBH studies and implicated both calcium and oxidized protein thiols (mixed disulfides) in the tBH damage mechanism and lipid peroxidation was shown to be associated with tBH-induced membrane leakiness, although detailed cause-and-effect relationships were not resolved. We will now seek to achieve a better understanding of functional impairment and oxidation products than had been possible in the earlier tBH studies, exploiting improved free radical detection and bioenergetics assays, based on our ESR technology. The HBO studies will parallel those with tBH and will also include quantitation of intra- and extra-mitochondrial superoxide production. The parallel tBH/HBO studies will include determinations of oxidized thiol species, functional inactivation (loss of membrane potential, ion efflux), lipid peroxidation, and analysis of the roles of calcium and free iron. A new assay for mixed disulfides will be perfected in the protein model system and then applied to mitochondria. Nutritional deficiencies in selenium or vitamin E, to decrease the effects of hydroperoxide metabolism and lipid peroxidation, together with assays of volatile gases associated with Fenton chemistry and lipid peroxidation will be used to attempt to gauge the importance of these oxidative damage processes in mitochondrial HBO effects. The applicability of the same volatile gas assays to a rat model will be exploited to seek preliminary correlations between some of the in vitro and in vivo effects of HBO treatment.